The object of the invention is to provide a composition for alleviation of uremic symptoms, which principle has a specific NPN compounds degrading effect, by utilizing non-pathogenic soil bacteria introduced into the intestine of persons suffering from uremia, whereby a biodegradation of NPN compounds possibly followed by a biosynthesis is made possible. Furthermore, the formation and concentration of nitrogen containing "waste products" in the organism is reduced.
To demonstrate the frequency of renal diseases, the following may be cited: in the forewords of The Manual of Artificial Organs, Vol. 1: The Artificial Kidney (author Y, Nose, Mosby Co., Saint Louis 1969) W. J. Kolff writes e.g. "the more we are worried and unhappy over the fact that only 1.5% of the 40,000 persons in need are helped by kidney transplantation or the artificial kidney, the more we search for a solution to the cruel problem caused by chronic renal failure". W. J. Kolff writes further in the forewords of "Hamodialyse und Peritonealdialyse" (authors P. Dittrich et al., Springer Verlag, Berlin Heidelberg New York 1969) that two high level committees, the Gotschalk-committee and the Burton-committee, have presented remarkably objective reports in the United States. If all those 40,000 patients which need dialysis or kidney transplantation were to be treated, the costs in the United States would be 600,000,000 dollars per year. But as stated earlier only one and a half percent are treated and rest are simply left to die. It is to be remembered that the number of lethal automobile accidents in the United States was 57,000 in the year 1966. This number is comparable with the number of uremia patients per year who are in a final stage of the illness and who would be considered for kidney transplantation.
Regarding uremic syndrome the following manuals are referred to: "Hamodialyse and Peritonealdialyse", P. Dittrich et al., Springer Verlag, Berlin Heidelberg New York, 1969; "The Kidney", A. Golden and J. F. Maher, Williams and Wilkins Co., Baltimore, 1971; "Uremia: Progress in Pathophysiology and Treatment", J. P. Merill and C. L. Hampers, Grune and Stratton, New York, 1971.
The following terms are used interchangeably herein: Uremic syndrom = chronic progressive irreversible renal failure = irreversible functional disorder of the kidneys.
Basic pathogenesis: for the time being unknown, only the symptoms are known. It is a complex symptom which often relates to increased metabolite retention values in the body fluids. There is often little conformity between chemical determinations and clinical observations. It is not known which of the numerous NPN compounds measured in the blood and other body fluids are toxic and/or responsible for the uremic symptoms. Guanidine is one of the NPN:s which has been considered "toxic". The blood-fluid limit might change into uremia. Urea possibly influences the migration of other NPN compounds through the physiological membranes and possibly plays an important role in the diffusion of more toxic substances into the urine.
Severe uremia brings forth general incapability. The patients are weak in conjunction with having other symptoms, as for example sickness, anorexia, vomiting, loss of weight, incapability of mental concentration, hypertony including its different consequences which often follow from renal failure and add to the symptoms. In many patients, different degrees and types of anemia and/or protein and/or salt exhaustion might influence the cell nutrition. All changes cannot be related to the retention of urea and other NPN compounds in the organism. Water intoxication, non-equilibrium in the K- and Na-distribution could be important. In addition, neuronal changes appear. Depression of the central nervous system and neuromuscular irritability have been declared as effects of, e.g., guanidine. The heart and pericardial structures might be influenced. The lungs might show signs of uremic pneumonitis. Pathological changes in the gastrointestinal tract are frequent. Certain serum enzymes might undergo changes in uremic patients. Anemia develops because of bone marrow suppression and/or hemolysis.
In the present therapy of uremia, it is noted that it includes kidney transplantation and dialysis, such as, hemodialysis, peritoneal dialysis and different enterodialysises or gastrointestinal drainage, respectively, with or without diet of energy rich, high quality nutrition. Kidney transplantation can for different reasons be carried out on only a limited number of kidney patients. There is for example a shortage of suitable donors. Basic immunologic principles are still unsolved. In some cases the transplanted kidney will be affected in the same way as the original one.
The method mostly used in connection with cronic renal insufficiency is the hemodialysis. The blood of the kidney patient is dialyzed, e.g. once a week or every second or every third day, year in, year out. Large amounts of accumulated NPN compounds (uremic "waste substances" ) are removed from the blood by hemodialysis. Thereby the blood-urea concentration for example during a hemodialysis lasting about 8 hours, decreases about 50% compared to the initial value. But the NPN values in the blood increase rapidly again, e.g., the serum-creatine value reaches its initial value within about 24 hours. Peritoneal dialysis is principally almost as effective as hemodialysis; it must also be carried out repeatedly. Its usefulness is limited and it cannot be used continuously.
Certain amounts of urea may be removed also by enterodialysis or gastrointestinal drainage. These methods cannot be repeated several times. Furthermore, NPN compounds, such as, e.g. creatinine, are not removed from the blood.
It may be concluded that the human body seems to be lavish in its excretion of NPN compounds through the kidneys. Actually huge amounts leave the organism, as, for example, urea 25 to 35 g daily (= 8 to 12 kg yearly), uric acid 0.6 to 0.9 g daily (= 0.2 to 0.3 kg yearly) and creatinine 1 to 2 g daily (= 0.3 to 0.7 kg yearly).
In ruminant egg, proteins are synthetized directly from hydrolyzed urea by microorganisms. A diet containing small amounts of nitrogen and large amounts of carbohydrates favours the protein synthesis. The re-use of "waste nitrogen" presumably lessens the animal's dependence on environmental protein sources. Sheep on a low protein diet are able to re-use at least 50% of the daily produced endogenous urea instead of excreting it. Experiments on ruminants show furthermore that creatinine is metabolized by rumen bacteria with the production of ammonia: creatine and creatinine is degraded by rumen microorganisms to urea and sarcosine. Part of the ammonia (NH.sub.3) formed in the rumen is used by the bacteria for synthesis of cell constituents. Part is absorbed through the ruminal wall into the portal blood stream. Part leaves the rumen into the lower portions of the intestine. The capability of the ruminal bacteria to use ammonia is dependant on the simultaneous availability of other nutrients required for the synthesis of their cellular constituents. Especially important are suitable carbon and energy sources. The presence of inorganic ions for the rumen-urease activity is critical. The results show that the urease activity is stimulated by the presence of Mn, Mg, Ca, Sr and Ba. Mn is the most effective. The ruminal bacteria produce and require B vitamins. The branched volatile fatty acids apparently play an important role in providing a carbon skeleton for the biosynthesis of those amino acids which are not synthesized by certain rumen bacteria. Besides the degradation of urea, different microorganisms, because of the enzyme system contained therein, are capable of degrading creatine, creatinine, uric acid and so on, that is, those NPN compounds which accumulate in the organism of uremic patients. The formation of some of the enzyme systems in the organism depends on the presence of the substrate in the medium. The enzymes responsible for the oxidation of creatinine, lactose or malonic acid, are "totally adaptive", i.e. they are not formed in the absence of the substrate in question. The enzymes responsible for the degradation of several other substances, as for example uric acid, amino acids, lactic acid are "partly adaptive", i.e. their formation occurs to a certain degree in the absence of the substrate from the medium and is highly increased by its addition to the medium. Other enzyme systems, as, for example, those responsible for the oxidation of glucose or glycerol are constitutive.
In humans, the following aspect on the NPN metabolism may be considered. Urea hydrolysis takes place in the gastrointestinal tract and depends exclusively on bacteria. The digestion in ruminants stays in a definite close connection with that in monogastric animals. In adults, the gastrointestinal tract/the intestine is inhabited by several species, types and strains of microorganisms. The amount of microorganisms in the human (large) intestine is enormous: thus one milligram of feces contains about 150,000,000 microorganisms, and bacteria form about 2/3 of the dry feces. A few days after birth the intestine flora starts to develop and the blood-coagulation time becomes normal as a result of the bacterial synthesis of vitamin K in the intestine. In adults, intestinal bacteria synthetize numerous vital substances. In patients suffering from chronic progressive irreversible renal failure, e.g., enterodialysis results in a passive crossing of the mucous membranes following differences between gradients, e.g. urea, and others pass by means of active forces (part of the electrolytes). From the point of view of the invention, it is important that the movement of urea takes place rapidly.
Concerning uremic patients, the utilization of urea-nitrogen for the protein synthesis has been discussed. The consequences of irreversible renal failure are, as known, directly related to the amount/degree of protein catabolism. When this can be minimized not only is the accumulation and retention, respectively, of NPN compounds delayed and decreased, but also, the load of K, P and so on, and their disappearance are prevented. One of the goals in the treatment of irreversible renal failure is to keep the nitrogen balance on the lowest possible intake of low-value proteins and/or amino acids. Some results have been obtained in the treatment of uremic patients on a corresponding diet, presumably because the nitrogen balance is kept in equilibrium by the intake of essential amino acids and high-value proteins. Investigations of the urea metabolism during low protein intake have shown that e.g. nitrogen from urea, apparently via bacterial hydrolysis to ammonia, may be used for protein synthesis. Urea production is reduced on a low protein diet but the urea degradation is sustained. It has further been shown that if the nutritional problem is tackled by means of a suitable diet in the case of renal failure, it is likely that at least some of the clinical problems, such as, acidosis, hyperkalemia, and phosphate retention will disappear, as well as some of the symptoms of uremia which probably are caused by urea itself. Urea has been administered to children during the investigation of certain problems in order to show that children are able to utilize urea and some other simpler nitrogen containing substances. It has also been suggested to "cycle backwards" the urea metabolism in the organism under favourable circumstances. It has further been suggested that the urea production in patients with renal failure is dependent on the protein intake down to a value of 20 g/day. A low protein diet consisting of so called natural foods suitable for uremic patients usually contains too few calories, i.e., energy. On the other hand, the addition of a purely of carbohydrate containing caloric supplement reduces urea production to a significant degree. In most kidney patients treated in this way, 20 to 80 percent of the urea formed is metabolized extra-renally. A literature study relating to the production and extra-renal (outside the kidneys) metabolism of urea in kidney patients treated with diet and dialysis, show that a substantial extra-renal urea degradation can occur in these patients, and further, that a reduction of the urea pool in the organism by dialysis, reduces urea degradation and furthermore that increased urea degradation in the presence of high body fluid urea concentration may be an effect of adaptive enzymes or represent reversal of the urea cycle.
In the human environment, there are microorganisms, or soil bacteria which are able to biodegrade or decompose, respectively, those NPN compounds which also accumulate in the body of kidney patients. Experiments with some of the soil bacteria have shown that the bacterial enzymes are easily adaptable. Furthermore, the optimal pH value and growth temperature required for maximum biodegradation of e.g., creatinine are similar to the values inside the rumen. The enzyme systems of the soil bacteria are relatively specific for known creatinine and creatine. Thus, for example, creatine biodegrading soil bacteria, during the degradation, refuse compounds closely related to creatinine, or affect them only slowly. It should also be noted that most of the soil bacteria are usually non-pathogenic for humans when administered per os, i.e., when introduced via the digestive canal into the organism.
The microbial flora in the human digestion system may be influenced in many different ways. Even implantation, enrichment or replacement of a number of microorganisms in the gastrointestinal tract is possible. Purposeful administration of "trained" and "untrained", respectively, microorganisms has already been carried out as a therapeutical method. The administered microorganisms do however in the therapeutical treatment relatively rapidly disappear from the gastrointestinal tract after interruption of the treatment. On the other hand, microorganisms might live longer in the gastrointestinal tract provided that specific substrates together with other vital building components are present.
It has now surprisingly been found that uremic symptoms may be removed in patients by administering per os, a medical preparation that comprises -- and this is the invention -- a basic component of a system derived from non-pathogenic soil bacteria having the characteristics of degrading non-protein nitrogen compounds (NPN) and being active in the gastrointestinal tract. The basic component prepared by utilizing soil bacteria contains specific, constitutive enzyme systems having the characteristics of degrading NPN compounds.
The soil bacteria may also be administered in lyophilized form.
The composition contains as urea degrading soil microorganisms, Serratia, species and as creatinine degrading soil bacteria,
(a) non-fluorescent Pseudomonas or PA0 (b) Rhizobium or PA0 (c) Agrobacterium or PA0 (d) Corynebacterium ureafaciens or PA0 (e) Arthrobacter ureafaciens or PA0 (f) Escherichia coli or PA0 (g) Pseudomonas aeruginosa; PA0 (a) non-fluorescent species of Pseudomonas or PA0 (b) Bacillus subtilis or PA0 (c) Bacillus fastidosus or PA0 (d) Micrococcus dentrificans or PA0 (e) Mycobacterium phlei or PA0 (f) Aerobacter aerogenes or PA0 (g) Fusarium moniliforme or PA0 (h) Histoplasma capsulata or PA0 (i) Penicillinum chrysogenum.
and as uric acid biodegrading soil microorganisms
The invention concerns also a method for the production of a composition for alleviation of uremic symptoms comprising selecting the species of urea degrading microorganism and cultivating this to determine that only one cell type grows in the culture medium, having the following composition: thiamine, pyridoxine, Ca-pantothenic acid, nicotinic acid, p-amino benzoic acid, each in an amount of 0.25 mg, 0.05 mg of folic acid, 1 .mu.g of B.sub.12 -vitamin, 0.5 of .mu.g biotine, 1.0 g of K.sub.2 HPO.sub.4, 1.5 g of NaH.sub.2 PO.sub.4 . 4H.sub.2 O, 0.05 g of MnCl.sub.2 . 4H.sub.2 O, 0.1 g of MgSO.sub.4 . 7H.sub.2 O, 0.01 g of FeSO.sub.4 . 7H.sub.2 O, 1.0 g of yeast extract and 10 g/1000 ml of urea, at a pH value of 7.0 to 7.5. If urea is used as a nitrogen source 5.0 g/1000 ml of glucose or glycerol is added, the bacteria culture is cultivated at 28.degree. C. in a fermentation vessel, the cell mass washed repeatedly with a buffer solution having a pH value of 7.2 and centrifugated, and thereafter added to 1 molar glucose solution at a ratio of 1:1 as stabilisator, whereafter the product is freezed in liquid nitrogen, thereafter lyophilized 48 to 72 hours at -50.degree. C. and 0.03 torr, while being protected from atmospheric oxygen, is exposed to an inert gas, such as argon, dried and the dried product formed into orally administrable preparations, such as, capsules or the like.
Numerous investigations both in vitro as well as in vivo, i.e., using a living object, have been carried out with the principle. The investigations in vivo comprised animal experiments (dogs) and clinical experiments (voluntary test persons as well as uremic patients). The effect and the method of preparation of the principle according to the invention is illustrated in following test results and examples.